Fluorescent dyes are widely used as tracers for localization of biological structures by fluorescence microscopy, for quantification of analytes by fluorescence immunoassay, for flow cytometric analysis of cells, for measurement of physiological state of cells, for quantitative assays such as DNA sequencing and other applications [Y. Kanaoka, Angew Chem. Intl. Ed. Engl. 16, 137 (1977); I. Hemmila. Clin. Chem. 31, 359 (1985)]. Their primary advantages over other types of absorption dyes include visibility of the emission at a wavelength distinct from the excitation, the orders of magnitude greater detectability of fluorescence emission over light absorption, the generally low level of fluorescence background in most biological samples and the measurable intrinsic spectral properties of fluorescence polarization [M. E. Jolley, et al. Clin. Chem. 27, 1190 (1981)], lifetime and excited state energy transfer [U.S. Pat. No. 3,996,345].
For many applications that utilize fluorescent dyes as tracers, it is necessary to chemically react the dye with a biologically active ligand such as a cell, tissue, protein, antibody, enzyme, drug, hormone, nucleotide, nucleic acid, polysaccharide, lipid or other biomolecule to make a fluorescent ligand analog or with natural or synthetic polymers. With these synthetic probes the biomolecule frequently confers a specificity for a biochemical interaction that is to be observed and the fluorescent dye provides the method for detection and/or quantification of the interaction.
Chemically reactive synthetic fluorescent dyes have long been recognized as essential for following these interactions [A. H. Coons & M. H. Kaplan, J. Exp. Med. 91, 1 (1950); E. Soini & I. Hemmila. Clin. Chem. 25, 353 (1979)]. The fluorescent dyes currently used to prepare useful conjugates are limited to a relatively small number of aromatic structures. Reactive fluorescein and rhodamine derivatives have been by far the most commonly used fluorescent dyes for preparation of fluorescent conjugates that can be excited and detected at wavelengths longer than 480 nm. The spectral properties that are most useful in a dye are frequently limited by the principal excitation wavelengths available in the common light sources. Primary among these are the argon laser and the mercury arc lamp which are used in many of the most sensitive applications of fluorescent probes. Certain intrinsic properties of the known fluorescent dyes, that include absorbance and fluorescence yield, stability during illumination and sensitivity of the spectra to the environment, limit the suitability of current dyes, including the fluoresceins and rhodamines, particularly in quantitative applications. It is the object of this invention to provide improved fluorescent dyes. It is further an object of this invention to provide dyes with the chemical reactivity necessary for conjugation to a variety of the functional groups commonly found in biomolecules, drugs, and natural and synthetic polymers.
The useful dyes would have the following properties:
1. A high absorbance as measured by extinction coefficient and a high fluorescence quantum yield with a relatively narrow emission peak. PA1 2. High absorbance at the most intense emission lines of the common excitation sources such as the 488 nm and 514 nm lines of the argon ion laser, the 546 nm line of the mercury arc lamp and the 543 nm and 632 nm lines of the helium-neon laser. PA1 3. High solubility of the dye and its reactive derivatives in a variety of solvents to maximize the utility of the dye for modification of cells, biopolymers and other ligands of interest and a low tendency of the labelled ligands or biomolecules to aggregate. PA1 4. High stability of the dye to excitation light, enhancing the utility of the dye for quantitative measurements and permitting extended illumination time and higher lamp intensities for increased sensitivity. PA1 5. For quantitative measurements, low sensitivity of the emission intensity to properties of the solution is necessary so that the measured signal is proportional only to the absolute quantity of dye present and is relatively independent of environmental effects such as pH, viscosity and polarity. PA1 6. Suitability of the dye preparation of reactive derivatives of several different types which exhibit direct chemical reactivity toward a variety of the chemically reactive sites typically found in biomolecules and other ligands of interest. PA1 7. Intrinsically low biological activity or toxicity of the dye.
Coons and Kaplan in 1950 first prepared a chemically reactive isocyanate of fluorescein and later J. L. Riggs. et al. [Am. J. Pathol. 34, 1081 (1958)] introduced the more stable isothiocyanate analog of fluorescein. Fluorescein isothiocyanate (FITC) remains one of the most widely used tracers for fluorescent staining and immunoassay. Other reactive fluoresceins were prepared by Haugland [U.S. Pat. No. 4,213,904]. Virtually all fluorescence microscopes are equipped with excitation sources and filters optimized to excite and detect fluorescein emission. Due to the intense but discrete excitation of the argon laser at 488 nm which is strongly absorbed by the primary fluorescein absorption band (maximum at 492 nm), fluorescein has also become the primary dye for use in the techniques of flow cytometry [L. L. Lanier & M. R. Loken, J. Immunol. 132, 151 (1984)] and laser scanning microscopy. There is a recognized need for suitable fluorophores for applications in multi-color microscopy [H. Khalfan, et al., Histochem. J. 18, 497 (1986)], flow cytometry [Stryer, et al., U.S. Pat. No. 4,520,110; J. A. Titus, et al., J. Immunol. Methods 50, 193 (1982)], immunoassays [W. A. Staines, et al., J. Histochem. Cytochem. 36, 145 (1988)], and DNA sequencing [L. M. Smith, et al. Nature 321, 674 (1986)]. Multicolor fluorescence typically has employed fluorescein in combination with longer wavelength dyes such as the fluorescein derivatives eosin isothiocyanate [R. J. Cherry, FEBS Lett. 55, 1 (1975)], erythrosin isothiocyanate [C. J. Restall, Biochim. Biophys. Acta 670, 433 (1981)], chloro and methoxy substituted fluoresceins [U.S. Pat. No. 4,318,846] as well as rhodamine derivatives, such as tetramethylrhodamine isothiocyanate [J. A. Gourlay & J. R. Pemberton, Appl. Microbiol. 22, 459 (1971)], 5(6)-carboxytetramethylrhodamine, succinimidyl ester [P. L. Khanna, & E. F. Ullman, Anal. Biochem 108, 156 (1980)], X-rhodamine isothiocyanate [H. A. Crissman & J. A. Steinkemp, Cytometry 3, 84 (1982)], carboxy-X-rhodamine, succinimidyl ester [G. P. A. Vigers, et al., J. Cell Biol. 107, 1011 (1988)], Lissamine rhodamine B sulfonyl chloride [C. S. Chadwick, et al., Immunology 1, 315 (1958)] and Texas Red [J. A. Titus, R. P. Haugland, S. O. Sharrow, J. Immunol. Methods 50, 193 (1982)].
The primary advantages that have permitted fluorescein isothiocyanate and its conjugates to remain the standard for microscopy and fluorescence immunoassay are high absorptivity, a high quantum yield and general ease of conjugation to biomolecules. The only fluorescent tracers in common use that exceed the overall fluorescence yield of fluorescein on a molar basis are the phycobiliproteins [U.S. Pat. No. 4,520,110; V. T. Oi, et al. J. Cell Biol. 93, 981 (1982); M. N. Kronick & P. D. Grossman, Clin. Chem. 29, 1582 (1983)]. These require special methods for conjugation to biomolecules and in some cases such as fluorescence polarization immunoassays [M. E. Jolley, et al., Clin. Chem. 27, 1190 (1981)] have too high a molecular weight to be useful. Phycobiliproteins also have high susceptibility to photodegradation [J. C. White & L. Stryer, Analyt. Biochem. 161, 442 (1987)]. The only chemically reactive fluorophores with spectra similar to fluorescein that have been described are derived from the nitrofurazan structure [Soini and Hemilla (1979)] and the dipyrrometheneboron difluoride structure [Haugland and Kang, U.S. Pat. No. 4,774,339; Monsma, et al., J. Neurochem. 52, 1641 (1989)]. The nitrofurazan derivatives have much weaker absorptivity (less than 25,000 cm.sup.-1 M.sup.-1 at its peak at 468 NM versus 75,000 cm.sup.-1 M.sup.-1 for fluorescein at its peak near 490 NM) and virtually no fluorescence in aqueous solutions, where fluorescein is usually used, and where most applications in immunofluorescence exist. The dipyrrometheneboron difluoride dyes, while possessing high extinction coefficients and quantum yields, are less photostable and more hydrophobic than the subject fluorophores.
Despite their widespread acceptance as fluorescent tracers, derivatives of fluorescein and the recently introduced derivatives of dipyrrometheneboron difluoride have some deficiencies that preclude or make more difficult some useful applications. Primary is the strong tendency of the fluorophores to photobleach when illuminated by a strong excitation source such as the mercury or xenon arc lamps typically used in fluorescence microscopes. The photobleaching can result in a significant percentage of the fluorescence being lost within seconds of the onset of illumination. In fluorescence microscopy this results in loss of the image. In fluorescence assays, the loss of fluorescence with time makes quantification of results difficult and ultimately decreases the sensitivity of detection of the analyte. To a variable degree, extrinsic reagents including propyl gallate and p-phenylenediamine retard, but do not eliminate, the photobleaching. However, these anti-fade agents cannot be used in experiments with living cells, one of the major recent applications of fluorescent dyes and fluorescently labelled ligands. Additionally, fluorescein and its derivatives show a pH dependent absorption spectrum that decreases the fluorescence yield in solutions at physiological pH or below. Furthermore, in applications requiring simultaneous excitation of two or more dyes whose emission is to be quantified separately, it is often more desirable to use the 514 nm line of the argon ion laser, since the extinction coefficient of the commonly used long wavelength dyes such as tetramethylrhodamine, rhodamine B and Texas Red is considerably higher at 514 nm than at 488. The absorption intensity of fluorescein at 514 nm is less than 10% of its maximum intensity at 492 nm and shows proportionally lower fluorescence intensity when excited at 514 nm.
In addition to the 488 nm and 514 nm lines of the argon ion laser, other significant, intense excitation wavelengths available from common sources are the 532 nm line of the Nd:YAG laser and the 546 nm line of the mercury arc lamp as well as the 543 nm and 632 nm lines of helium neon lasers. Since the sensitivity of detection of fluorescent ligands is proportional to the product of the lamp intensity, the absorbance and the quantum yield, dyes having significant absorbance and fluorescence at these longer wavelengths, such as some of the dyes which are the subject of this invention, are also useful. By far the most common reactive dye in current use that can be excited at these wavelengths has been tetramethylrhodamine, commonly used as its isothiocyanate derivative, TRITC.
Tetramethylrhodamine, while pH insensitive and relatively photostable, possesses several deficiencies which limit its utility as a fluorescent label. Primary among these is the relatively low quantum yield of the dye in aqueous solution. While the quantum yield of tetramethylrhodamine in alcohol is above 0.90, the quantum yield in water drops to approximately 0.23, which decreases the sensitivity of detection concomitantly. Protein conjugates of tetramethylrhodamine show a further decrease in fluorescence quantum yield and exhibit complex absorption behavior resulting from the tendency of rhodamine derivatives to aggregate in aqueous solution [O. Valdes-Aguilera & D. C. Neckers, Acc. Chem. Res. 22, 171 (1989)]. Another drawback of tetramethylrhodamine is the high sensitivity of emission to solvent polarity and viscosity. Additionally, the low water solubility of reactive forms of the dye and its conjugates present difficulties for the preparation and use of rhodamine labelled probes.
Clearly there is a need and opportunity for more fluorescent and more photostable reactive fluorophores. The base for the new chemically reactive fluorophores of this invention is a rhodol compound. While a number of substituted and unsubstituted derivatives of the parent heterocyclic compound have been described [I. S. Ioffe & V. F. Otten, J. Org. Chem. USSR 1, 326-336 (1965); G. A. Reynolds, U.S. Pat. No. 3,932,415; L. G. Lee, et al., Cytometry 10, 151 (1989)], their research did not provide methods whereby the fluorophores could be chemically reacted with ligands.